Methods of treating diseases which are mediated by cutaneous lymphocyte antigen positive cells

ABSTRACT

The present invention relates to methods of treating patients suffering from itching and puritis mediated by cutaneous lymphocyte antigen positive T cell. In particular, diseases or disorders including contact dermatitis, drug induced delayed type cutaneous allergic reactions, toxic epidermal necrolysis, cutaneous T cell lymphoma, bullous pemphigoid, alopecia aereata, vitiligo, acne rosacea, prurigo nodularis, and herpes simplex virus, or combination thereof will benefit from the administration of an IL-31 antagonist. The invention also includes methods of predicting a therapeutically responsive patient population.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/094,482, filed Apr. 26, 2011, which is a continuation of U.S.patent application Ser. No. 12/497,383, filed Jul. 2, 2009, nowabandoned, which is a continuation of U.S. patent application Ser. No.11/353,427, filed Feb. 14, 2006, now abandoned, which claims the benefitof U.S. Provisional Application Ser. No. 60/653,114, filed Feb. 14,2005, U.S. Provisional Application Ser. No. 60/716,762, filed Sep. 13,2005, and U.S. Provisional Application Ser. No. 60/749,952, filed Dec.13, 2005, all of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

The skin plays an important role in the immune system and consists oflayers. Circulating T lymphocytes migrate to the skin under normal andinflammatory conditions. The cutaneous lymphocyte antigen (CLA) isconsidered a homing receptor for T cells with tropism for the skin.Santamaria-Babi, L., Eur. J. Dermatol. 14:13-18, 2004. CLA is acarbohydrate structure which is expressed on memory T cells as anepitope of the single cell-surface protein named P-selectin glycoproteinligand-1 (PSGL-1) and facilitates binding of T cells to E-selectin, aninducible adhesion molecule expressed on vascular endothelium. SeeFuhlbrigge R C, et al., Nature 1997; 389:978-81.

Several diseases of the skin are known to express high levels of CLA+ Tcells, including atopic dermatitis, contact dermatitis, drug-inducedallergic reactions, skin-tropic viruses and viral associated pruritis,vitiligo, cutaneous T cell lymphoma, alopecia aerata, acne rosacea, acnevulgaris, prurigo nodularis, and bullous pemphigoid. There is a need totreat such skin T cell mediated diseases.

The demonstrated in vivo activities of cytokines illustrate the enormousclinical potential of, and need for, other cytokines, cytokine agonists,and cytokine antagonists. The present invention addresses these needs byproviding a method of treating such diseases by interfering with theactions of IL-31, a newly identified cytokine. IL-31, whenover-expressed in mice, results initching and dermatitis-like symptoms.Both skin-homing T cells and epidermal keratinocytes have beenimplicated in the pathology of skin diseases in humans.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

SUMMARY OF THE INVENTION

Within one aspect, the invention provides a method of treating diseasedskin comprising administering an antagonist molecule to a mammal withthe diseased skin wherein the diseased skin is characterized bycutaneous lymphocyte antigen positive T cells and the antagonistmolecule specifically binds to the polypeptide comprising the amino acidsequence as shown in SEQ ID NO:2 or SEQ ID NO: 4, and wherebyadministration of the antagonist molecule improves, prevents, inhibitsor reduces the diseased skin. Within an embodiment, the patient has askin disorder selected from Contact dermatitis, Drug induced delayedtype cutaneous allergic reactions, Toxic epidermal necrolysis, CutaneousT cell Lymphoma, Bullous pemphigoid, Alopecia aereata, Vitiligo, AcneRosacea, Prurigo nodularis, and Herpes simplex virus. Within a furtherembodiment, the mammal is a human. Within another embodiment, theantagonist is an antibody or antibody fragment. Within a furtherembodiment the antagonist molecule specifically binds to the polypeptidecomprising the amino acid sequence as shown in SEQ ID NO:2. Withinanother embodiment, the diseased skin is pruritic.

Within another aspect, the invention provides a method for treatingpruritis comprising administering an antagonist molecule to a mammalwith the pruritis wherein the pruritis is characterized by cutaneouslymphocyte antigen positive T cells and wherein the antagonist moleculespecifically binds to the polypeptide having the amino acid sequence asshown in SEQ ID NO:2 or in SEQ ID NO: 4, and whereby administration ofthe antagonist molecule improves, prevents, inhibits or reduces thepruritis. Within an embodiment, the pruritis is associated with a skindisorder selected from Contact dermatitis, Drug induced delayed typecutaneous allergic reactions, Toxic epidermal necrolysis, Cutaneous Tcell Lymphoma, Bullous pemphigoid, Alopecia aereata, Vitiligo, AcneRosacea, Prurigo nodularis, and Herpes simplex virus. Within an furtherembodiment, the mammal is a human. Within a further embodiment, theantagonist is an antibody or antibody fragment. Within a furtherembodiment, the antagonist molecule specifically binds to thepolypeptide comprising the amino acid sequence as shown in SEQ ID NO:2.

Within another aspect, the invention provides a method for predictingtherapeutic response to an IL-31 antagonist in an individual in need ofIL-31 antagonist therapy comprising obtaining a biological sample fromthe patient, isolating circulating cutaneous lymphocyte positive T cellsfrom the biological sample, and detecting IL-31 production from theisolated cutaneous lymphocyte positive T cells. Within an embodiment,the IL-31 is detected by specifically binding to an IL-31 antagonist.Within a further embodiment, the IL-31 antagonist is an anti-IL-31antibody or antibody fragment. Within another embodiment, the antagonistmolecule specifically binds to the polypeptide comprising the amino acidsequence as shown in SEQ ID NO:2. Within another embodiment, theindividual in need of IL-31 antagonist therapy has a skin disorderselected from Contact dermatitis, Drug induced delayed type cutaneousallergic reactions, Toxic epidermal necrolysis, Cutaneous T cellLymphoma, Bullous pemphigoid, Alopecia aereata, Vitiligo, Acne Rosacea,Prurigo nodularis, and Herpes simplex virus. Within another embodiment,the method comprises the additional step of stimulating or activatingthe cutaneous lymphocyte antigen positive T cells. Within a furtherembodiment, the IL-31 is detected by specifically binding to an IL-31antagonist. Within another embodiment, the IL-31 antagonist molecule isan anti-IL-31 antibody or antibody fragment. Within a furtherembodiment, the antagonist molecule specifically binds to thepolypeptide comprising the amino acid sequence as shown in SEQ ID NO:2.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a polynucleotide that has a contiguous stretchof identical or complementary sequence to another polynucleotide.Contiguous sequences are said to “overlap” a given stretch ofpolynucleotide sequence either in their entirety or along a partialstretch of the polynucleotide.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e., greater than 95% pure,more preferably greater than 99% pure. When used in this context, theterm “isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “neoplastic”, when referring to cells, indicates cellsundergoing new and abnormal proliferation, particularly in a tissuewhere in the proliferation is uncontrolled and progressive, resulting ina neoplasm. The neoplastic cells can be either malignant, i.e., invasiveand metastatic, or benign.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention provides novel methods of using IL-31polynucleotides, polypeptides, and antagonists in detection, diagnosis,and treatment of diseases, in particular, diseases that are mediated bycutaneous lymphocyte antigen (CLA) positive T cells. The presentinvention is based in part upon the discovery that a previouslyidentified cytokine, IL-31 is expressed by skin-homing T cells, but notgut-homing T cells.

IL-31 is a recently discovered protein having the structure of afour-helical-bundle cytokine. This cytokine was previously identified asIL-31 and is fully described in U.S. patent application Ser. No.10/352,554, filed Jan. 21, 2003. See published U.S. Patent ApplicationNo. 2003-0224487, and PCT application WO 03/060090, all hereinincorporated by reference. See also, Dillon, et al., Nature Immunol.5:752-760, 2004. IL-31 is a ligand with high specificity for thereceptor IL-31RA and at least one additional subunit comprisingOncostatinM receptor beta (OSMRbeta). The native polynucleotide andpolypeptide sequences for human IL-31 are shown in SEQ ID NOs: 1 and 2,respectively. The native polynucleotide and polypeptide sequences formouse IL-31 are shown in SEQ ID NOs: 3 and 4, respectively. The nativepolynucleotide and polypeptide sequences for human IL-31RA are shown inSEQ ID NOs: 5 and 6, respectively. The native polynucleotide andpolypeptide sequences for mouse IL-31RA are shown in SEQ ID NOs: 7 and8, respectively. The native polynucleotide and polypeptide sequences forhuman OSMRbeta are shown in SEQ ID NOs: 9 and 10, respectively.

The secretory signal sequence of IL-31 is comprised of amino acidresidues 1 (Met) to 23 (Ala), and the mature polypeptide is comprised ofamino acid residues 24 (Ser) to 164 (Thr) as shown in SEQ ID NO:2.Further N-terminal sequencing analysis of purified IL-31 from 293T cellsshowed an N-terminus at residue 27 (Leu) as shown in SEQ ID NO:2, withthe mature polypeptide comprised of amino acid residues 27 (Leu) to 164(Thr) as shown in SEQ ID NO:2.

As used herein the term, IL-31 means Zcytor17lig, and IL-31RA meansZcytor17, as used in U.S. patent publication number 20030224487 (hereinincorporated by reference), as shown above. The heterodimeric receptorfor IL-31 was also described in 2003-0096339 (also incorportated hereinby reference) as zcytor17 (HUGO name, IL-31RA) which form a heterodimerwith at least one additional subunit comprising OncostatinM receptorbeta (OSMRbeta).

Both skin-homing T cells and epidermal kerationcytes have beenimplicated in the pathology of skin diseases in humans. As shown herein,IL-31 mRNA and protein expression is restricted to the skin-homing CLA+T cell population in both atopic dermatitis (AD) patients and normalindividuals, while analysis of the receptor for IL-31, IL-31RA, byimmunohistochemistry (IHC) suggests slightly higher levels of IL-31RAexpression on skin keratinocytes in skin biopsies from AD suffererscompared to normal individuals.

When over-expressed in mice, IL-31 results in pruritus and thedevelopment of skin dermatitis resembling human atopic dermatitis (AD).Immunohistochemistry (IHC) studies shown herein show that IL-3 RAprotein was expressed by skin keratinocytes and infiltrating macrophagesin skin biopsies from AD patients. Comparisons between AD patients andnormal individuals suggested that IL-31RA was expressed at higher levelson epidermal keratinocytes in the AD samples. Skin cell infiltrates,which were present at greater numbers in skin of AD patients compared tonormal individuals, expressed IL-31 mRNA. Histomorphometric analysis ofthese cells suggested a lymphocytic lineage with the majority of cellsstaining positive for cutaneous lymphocyte antigen (CLA) and CD3,demonstrating that skin-homing T cells in skin express IL-31 mRNA. Uponanalysis of peripheral blood T cells for IL-31, IL-31 mRNA and proteinexpression is largely restricted to CD45RO+ CLA+ skin-homing T cells inAD and normal volunteers. Moreover, circulating CLA+ T cells from ADpatients are capable of producing higher levels of IL-31 compared toCLA+ T cells from normal individuals, though there is large variabilitybetween patient samples. These results provide strong evidence thatIL-31 expression may contribute to the development of AD skininflammation and pruritus.

As shown herein, IL-31 is produced both locally in the skin and by skininfiltrating cells. Local production of cytokines in tissues by T cellsis thought to be a key mechanism for disease pathogenesis in AD andincreased numbers of T cells both in circulation and in skin is thoughtto correlate with disease.

Although both AD patients and normal controls have circulating CLA+ Tcells that express IL-31 upon activation, CLA+ T cells from AD patientsare reported to exist in a more activated state compared to cells fromnormal individuals. See Akdis M, J Immunol 159:4611-4619, 1997.Consequently, the threshold of stimulation required for the productionof IL-31 by CLA+ T cells may differ between dermatitis patients andcontrol subjects. As shown herein, circulating CLA+ T cells from ADpatients after 24 hours of stimulation with sub-optimal concentrationsof anti-CD3 in the absence of anti-CD28 have the capacity to producehigher levels of IL-31 compared to cells from normal individuals. Due tothe variability in IL-31 levels produced by CLA+ T cells from individualAD patients, there was no significant difference in the average IL-31production from circulating CLA+ T cells of AD and normal individuals.Nevertheless, since more CLA+ T cells are localized in skin of ADpatients, as compared to normal individuals, there is an increasedpotential for IL-31 activity in the AD skin micro-environment.

Example 8 demonstrates that circulating CLA+ T cells from some ADpatients produce higher levels of IL-31 compared to cells from normalindividuals. The detection of IL-31 in patients of such a subpopulationusing the bioassay provided herein, or with any assay that detects IL-31produced by circulating T cells in the blood, may be useful to determineif an IL-31 antagonist will be useful as treatment for diseases whereinthe presence of IL-31 causes inflammation.

A cell line that is dependent on the OSMRbeta and IL-31RA linked pathwayfor survival and growth in the absence of other growth factors can beused to measure the activity of IL-31. Such growth factor-dependent celllines include BaF3, FDC-P1, and MO7e. For information on the BaF3 cellline, see Palacios and Steinmetz, (Cell 41: 727-734, 1985) andMathey-Prevot et al., (Mol. Cell. Biol. 6: 4133-4135, 1986). Forinformation on the FDC-P1cell line, see Hapel et al. (Blood 64: 786-790,1984). For information on the MO7e cell line, see Kiss et al., (Leukemia7: 235-240, 1993).

The amino acid sequence for the OSMR, and IL-31RA receptors indicatedthat the encoded receptors belonged to the Class I cytokine receptorsubfamily that includes, but is not limited to, the receptors for IL-2,IL-4, IL-7, Lif, IL-12, IL-15, EPO, TPO, GM-CSF and G-CSF (for a reviewsee, Cosman, “The Hematopoietin Receptor Superfamily” in Cytokine 5(2):95-106, 1993). The IL-31RA receptor is fully described in PCT PatentApplication No. US01/20484 (WIPO publication No. WO 02/00721). Analysisof the tissue distribution of the mRNA of the IL-31RA receptor revealedexpression in activated CD4+ and CD8+ T-cell subsets, CD14+ monocytes,and weaker expression in CD19+ B-cells. Moreover, the mRNA was presentin both resting or activated monocytic cell lines THP-1 (ATCC No.TIB-202), U937 (ATCC No. CRL-1593.2) and HL60 (ATCC No. CCL-240).

IL-31 is considered a four-alpha helix structure. Referring to the humanIL-31 amino acid sequence shown in SEQ ID NO:2, the IL-31 helix A isdefined by amino acid residues 38-52; helix B by amino acid residues83-98; helix C by amino acid residues 104-117; and helix D by amino acidresidues 137-152, and the conserved cysteine residues within IL-31correspond to amino acid residues 72, 133, and 147 of SEQ ID NO:2; and74, 137, and 151 of SEQ ID NO:8 described herein. Also highly conservedin the IL-31 is the Glu residue as shown in SEQ ID NO:2 at residue 43.

The polynucleotide sequence for the mouse ortholog of IL-31 has beenidentified and is shown in SEQ ID NO:3 and the corresponding amino acidsequence shown in SEQ ID NO:4. For the IL-31 mouse cytokine amino acidsequence of SEQ ID NO: 4, helix A is defined by amino acid residues38-52; helix B by amino acid residues 85-98; helix C by amino acidresidues 104-118; and helix D by amino acid residues 141-157. Maturesequence for the mouse IL-31 putatively begins at Met₁, as shown in SEQID NO:4, which corresponds to Met₁, as shown in SEQ ID NO:2, in thehuman sequence. Tissue analysis revealed that expression of mouse IL-31is found in testis, brain, CD90+ cells, prostate cells, salivary glandand skin. Further N-terminal sequencing analysis of purified IL-31 from293T cells showed an N-terminus at residue 31 (Ala) as shown in SEQ IDNO:4 with the mature polypeptide comprising amino acid residues 31 (Ala)to 163 (Cys).

IL-31 is located at the 12q24.31 region of chromosome 12. Thus, thepresent invention also provides reagents which will find use indiagnostic applications. For example, the IL-31 gene, a probe comprisingIL-31 DNA or RNA or a subsequence thereof, can be used to determine ifthe IL-31 gene is present on a human chromosome, such as chromosome 12,or if a gene mutation has occurred. Detectable chromosomal aberrationsat the IL-31 gene locus include, but are not limited to, aneuploidy,gene copy number changes, loss of heterozygosity (LOH), translocations,insertions, deletions, restriction site changes and rearrangements. Suchaberrations can be detected using polynucleotides of the presentinvention by employing molecular genetic techniques, such as restrictionfragment length polymorphism (RFLP) analysis, short tandem repeat (STR)analysis employing PCR techniques, and other genetic linkage analysistechniques known in the art (Sambrook et al., ibid.; Ausubel et. al.,ibid.; Marian, Chest 108:255-65, 1995). Detection of chromosomalaberrations may be particularly important for diseases with a highcorrelation of cutaneous lymphocyte antigen. Thus, the present inventionincludes methods of detecting changes in the IL-31 gene, including up ordown regulations thereof.

The proteins of the present invention (or polypeptide fragments thereof)can be joined to other bioactive molecules, particularly othercytokines, to provide multi-functional molecules. For example, one ormore helices from IL-31 can be joined to other cytokines to enhancetheir biological properties or efficiency of production.

The present invention also provides the use of detecting polypeptidefragments or peptides comprising an epitope-bearing portion of a IL-31polypeptide described herein in diseases mediated by CLA positive Tcells. Such fragments or peptides may comprise an “immunogenic epitope,”which is a part of a protein that elicits an antibody response when theentire protein is used as an immunogen. Immunogenic epitope-bearingpeptides can be identified using standard methods (see, for example,Geysen et al., Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodies(e.g., neutralizing antibodies) that bind with the polypeptidesdescribed herein. Hopp/Woods hydrophilicity profiles can be used todetermine regions that have the most antigenic potential (Hopp et al.,1981, ibid. and Hopp, 1986, ibid.). For example, in human IL-31,hydrophilic regions include amino acid residues 54-59 of SEQ ID NO:2,amino acid residues 129-134 of SEQ ID NO:2, amino acid residues 53-58 ofSEQ ID NO:2, amino acid residues 35-40 of SEQ ID NO:2, and amino acidresidues 33-38 of SEQ ID NO:2. For example, in mouse IL-31, hydrophilicregions include amino acid residues 34-39 of SEQ ID NO:4, amino acidresidues 46-51 of SEQ ID NO:4, amino acid residues 131-136 of SEQ IDNO:4, amino acid residues 158-163 of SEQ ID NO:4, and amino acidresidues 157-162 of SEQ ID NO:4.

Antigenic epitope-bearing peptides and polypeptides preferably containat least four to ten amino acids, at least ten to fourteen amino acids,or about fourteen to about thirty amino acids of SEQ ID NO:2 or SEQ IDNO:4. Such epitope-bearing peptides and polypeptides can be produced byfragmenting a IL-31 polypeptide, or by chemical peptide synthesis, asdescribed herein. Moreover, epitopes can be selected by phage display ofrandom peptide libraries (see, for example, Lane and Stephen, Curr.Opin. Immunol. 5:268 (1993); and Cortese et al., Curr. Opin. Biotechnol.7:616 (1996)). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBiology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al. (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

The IL-31 polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion polypeptides, can beproduced, purified and refolded by methods well-known in the art and asdescribed in published U.S. Patent Application No. 2003-0224487, and PCTapplication WO 03/060090. It is preferred to purify the polypeptides ofthe present invention to ≧80% purity, more preferably to ≧90% purity,even more preferably ≧95% purity, and particularly preferred is apharmaceutically pure state, that is greater than 99.9% pure withrespect to contaminating macromolecules, particularly other proteins andnucleic acids, and free of infectious and pyrogenic agents. Preferably,a purified polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin.

The present invention provides methods for using L-31 antagonists,including anti-IL-31 antibodies for reducing, inhibiting, or preventinginflammation in cell microenvironments where one or more cells in themicroenvironment is/are T cells that are positive for the cutaneouslymphocyte antigen. In addition the present invention provides methodsfor using IL-31 antagonists, including anti-IL-31 antibodies forreducing, inhibiting, or preventing itching and pruritis in cellmicroenvironments where one or more cells in the microenvironment is/areT cells that are positive for the cutaneous lymphocyte antigen.

Antibodies from an immune response generated by inoculation of an animalwith IL-31 antigens can be isolated and purified are know in the art andare described herein. Methods for preparing and isolating polyclonal andmonoclonal antibodies are well known in the art. See, for example,Current Protocols in Immunology, Cooligan, et al. (eds.), NationalInstitutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal HybridomaAntibodies: Techniques and Applications, CRC Press, Inc., Boca Raton,Fla., 1982.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication No.WO 98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination.

Antibodies are considered to be specifically binding if: 1) they exhibita threshold level of binding activity, and 2) they do not significantlycross-react with related polypeptide molecules. A threshold level ofbinding is determined if anti-IL-31 antibodies herein bind to a IL-31polypeptide, peptide or epitope with an affinity at least 10-foldgreater than the binding affinity to control (non-IL-31) polypeptide. Itis preferred that the antibodies exhibit a binding affinity (K_(a)) of10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸M⁻¹ or greater, and most preferably 10⁹ M⁻¹ or greater. The bindingaffinity of an antibody can be readily determined by one of ordinaryskill in the art, for example, by Scatchard analysis (Scatchard, G.,Ann. NY Acad. Sci. 51: 660-672, 1949).

Antibodies to IL-31 may be used for tagging cells that express IL-31;for isolating IL-31 by affinity purification; for diagnostic assays fordetermining circulating levels of IL-31 polypeptides; for detecting orquantitating soluble IL-31 as a marker of underlying pathology ordisease; in analytical methods employing FACS; for screening expressionlibraries; for generating anti-idiotypic antibodies; and as neutralizingantibodies or as antagonists to block IL-31 activity in vitro and invivo. Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to IL-31 or fragments thereof may be used in vitroto detect denatured IL-31 or fragments thereof in assays, for example,Western Blots or other assays known in the art.

Suitable detectable molecules may be directly or indirectly attached tothe polypeptide or antibody, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like. Suitable cytotoxic moleculesmay be directly or indirectly attached to the polypeptide or antibody,and include bacterial or plant toxins (for instance, diphtheria, toxin,saporin, Pseudomonas exotoxin, ricin, abrin and the like), as well astherapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90(either directly attached to the polypeptide or antibody, or indirectlyattached through means of a chelating moiety, for instance).Polypeptides or antibodies may also be conjugated to cytotoxic drugs,such as adriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

Binding polypeptides can also act as IL-31 “antagonists” to block IL-31binding and signal transduction in vitro and in vivo. These anti-IL-31binding polypeptides would be useful for inhibiting IL-31 activity orprotein-binding.

Both skin-homing T cells and epidermal keratinocytes have beenimplicated in the pathology of skin diseases in humans. As shown inExample 1 herein, of the T cell subsets, IL-31 mRNA and proteinexpression is restricted to the skin-homing CLA+ T cell population inhumans. As such, an antagonist to IL-31, including an antibody orreceptor antagonist will be useful in treating skin and epidermaldiseases which are mediated by CLA+ T cells. Such diseases include, forexample, atopic dermatitis, contact dermatitis, psoriasis, drug-inducedallergic reactions, skin-tropic viruses and viral associated pruritis,vitiligo, cutaneous T cell lymphoma, alopecia aerata, acne rosacea, acnevulgaris, prurigo nodularis, and bullous pemphigoid.

Atopic Dermatitis

Atopic dermatitis (AD) is a chronically relapsing inflammatory skindisease with a dramatically increasing incidence over the last decades.Clinically AD is characterized by highly pruritic often excoriatedplaques and papules that show a chronic relapsing course. The diagnosisof AD is mostly based on major and minor clinical findings. See HanifnJ. M., Arch Dermatol: 135, 1551 (1999). Histopathology revealsspongiosis, hyper and focal parakeratosis in acute lesions, whereasmarked epidermal hyperplasia with hyper and parakeratosis,acanthosis/hypergranulosis and perivascular infiltration of the dermiswith lymphocytes and abundant mast cells are the hallmarks of chroniclesions.

T cells play a central role in the initiation of local immune responsesin tissues and evidence suggests that skin-infiltrating T cells inparticular, may play a key role in the initiation and maintenance ofdisregulated immune responses in the skin. Approximately 90% ofinfiltrating T cells in cutaneous inflammatory sites express thecutaneous lymphocyte-associated Ag (CLA+) which binds E-selectin, aninducible adhesion molecule on endothelium (reviewed in Santamaria-BabiL. F., et al., Eur J Dermatol: 14, 13, (2004)). A significant increasein circulating CLA+ T cells among AD patients compared with controlindividuals has been documented (See Teraki Y., et al., Br J Dermatol:143, 373 (2000)), while others have demonstrated that memory CLA+ Tcells from AD patients preferentially respond to allergen extractcompared to the CLA−population (See Santamaria-Babi, L. F., et al., JExp Med: 181, 1935, (1995)). In humans, the pathogenesis of atopicdisorders of the skin have been associated with increases in CLA+ Tcells that express increased levels of Th-2-type cytokines like IL-5 andIL-13 9, 10. See Akdis M., et al., Eur J Immunol: 30, 3533 (2000); andHamid Q., et al., J Allergy Clin Immunol: 98, 225 (1996).

NC/Nga Mice spontaneously develop AD-like lesions that parallel human ADin many aspects, including clinical course and signs, histophathologyand immunopathology when housed in non-specified pathogen-free (non-SPF)conditions at around 6-8 weeks of age. In contrast, NC/Nga mice keptunder SPF conditions do not develop skin lesions. However, onset ofspontaneous skin lesions and scratching behaviour can be synchronized inNC/Nga mice housed in a SPF facility by weekly intradermal injection ofcrude dust mite antigen. See Matsuoka H., et al., Allergy: 58, 139(2003). Therefore, the development of AD in NC/Nga is a useful model forthe evaluation of novel therapeutics for the treatment of AD.

In addition to the NC/Nga model of spontaneous AD, epicutaneoussensitization of mice using OVA can also be used as a model to induceantigen-dependent epidermal and dermal thickening with a mononuclearinfiltrate in skin of sensitized mice. This usually coincides withelevated serum levels of total and specific IgE, however no skin barrierdysfunction or pruritus normally occurs in this model. See Spergel J.M., et al., J Clin Invest, 101: 1614, (1998). This protocol can bemodified in order to induce skin barrier disregulation and pruritis bysensitizing DO11.10 OVA TCR transgenic mice with OVA. Increasing thenumber of antigen-specific T cells that could recognize the sensitizingantigen may increase the level of inflammation in the skin to inducevisible scratching behaviour and lichenification/scaling of the skin.

Both the NC/Nga spontaneous AD model and the OVA epicutaneous DO11.10model are used to investigate expression of IL-31 and IL-31RA in AD. SeeExample 3.

An IL-31 neutralizing antagonist could be effective in inhibiting,reducing, minimizing or preventing atopic dermatitis reactions.

Contact Dermatitis

Allergic contact dermatitis is defined as a T cell mediated immunereaction to an antigen that comes into contact with the skin. The CLA+ Tcell population is considered to be involved in the initiation ofdermatitis since allergen dependent T cell responses are largelyconfined to the CLA+ population of cells (See Santamaria-Babi, L. F., etal., J Exp Med: 181, 1935, (1995)). Recent data has found that onlymemory (CD45RO+) CD4+ CLA+ and not CD8+ T cells proliferate and produceboth type-1 (IFN-γ) and type-2 (IL-5) cytokines in response to nickel, acommon contact hypersensitivity allergen. Furthermore, cells expressingCLA in combination with CD4, CD45RO (memory) or CD69 are increased afternickel-specific stimulation and express the chemokine receptors CXCR3,CCR4, CCR10 but not CCR6. See Moed H., et al., Br J Dermatol: 51, 32,(2004).

In animal models, it has been demonstrated that allergic contactdermatitis is T-cell dependent and that the allergic-responsive T cellsmigrate to the site of allergen application. See generally: Engeman T.M., et al., J Immunol: 164, 5207, (2000); Ferguson T. A. & Kupper T. S.J Immunol: 150, 1172, (1993); and Gorbachev A. V. & Fairchild R. L. CritRev Immunol: 21, 451 (2001). Since CLA+ T cells produce IL-31 and IL-31stimulation of skin keratinocytes can induce pro-inflammatorychemokines, IL-31 may be involved in the pathophysiology of contactdermatitis. See Example 2 for an in vivo model of contact dermatitis.

An IL-31 neutralizing antagonist could be effective in inhibiting,reducing, minimizing or preventing contact dermatitis reactions.

Drug-Induced Delayed Type Cutaneous Allergic Reactions

Drug-induced delayed type cutaneous allergic reactions are veryheterogeneous and may mirror many distinct pathophysiological events.See Brockow K., et al., Allergy: 57, 45 (2002). Immunological mechanismsinvolved in these reactions have been shown as either antibody or cellmediated. In immediate drug allergy an IgE-mediated antibody reactioncan be demonstrated by a positive skin prick and/or intradermal testafter 20 min, whereas non-immediate reactions to drugs can occur morethan one hour after last drug intake and are often T-cell mediated.Non-immediate T-cell mediated delayed type reactions can occur inpatients with adverse drug reactions to penicillins for example.Proliferative T cell responses to penicillins have been shown to berestricted to the memory (CD45RO+) CLA+ subpopulation of T cells frompenicillin allergic patients whereas the CD45RO+CLA−subset shows noproliferative response. See Blanca M., Leyva L., et al., Blood Cells MolDis: 31, 75 (2003). Delayed-type hypersensitivity (DTH) reactions can beartificially reproduced in mice, allowing assessment of factors that maybe involved in the initiation and perpetuation of the DTH response. AnIL-31 neutralizing antagonist could be effective in inhibiting,reducing, minimizing or preventing delayed type hypersensitivityreactions. See Example 4 for an in vivo model of DTH.

Toxic epidermal necrolysis (TEN) is a very rare but extremely severedrug reaction characterized by widespread apoptosis of epidermis withextensive blisters. Studies have shown that lymphocytes infiltrating theblister are CLA+ T cells and can exhibit cytotoxicity towards epidermalkeratinocytes. See Leyva L., et al., J Allergy Clin Immunol: 105, 157(2000); and Nassif A., Bensussan A., et al., J Allergy Clin Immunol:114, 1209 2004). A transgenic mouse system, whereby OVA is expressedunder the control of the keratin-5 (K5) promoter in the epidermal andhair follicular keratinocytes of mice, has been generated to establishan animal model for TEN. OVA specific CD8+ T cells, when adoptivelytransferred into K5-OVA mice, undergo activation and proliferation inthe skin-draining lymph nodes and target the skin of K5-OVA mice,resulting in development of skin lesions that are reminiscent of TEN.See Azukizawa H., et al., Eur J Immunol: 33, 1879 (2003). An IL-31neutralizing antagonist could be effective in inhibiting, reducing,minimizing or preventing TEN reactions.

Bullous Pemphigoid

Bullous pemphigoid is a subepidermal disorder which manifests assubepidermal blisters with a dermal infiltrate of neutrophils andeosinophils. Diagnosis is characterized by the presence ofantigen-specific antibodies against specific adhesion proteins of theepidermis and dermal-epidermal junction. See Jordon R. E., et al., JAMA:200, 751 (1967). Studies analyzing the role of T cells in thepathogenesis of bullous pemphigoid by analysis of PBL and skin blister Tcells have found a predominance of CLA+ T cells expressing increasedlevels of Th2-cytokines like IL-4 and IL-13. See Teraki Y., et al., JInvest Dermatol: 117, 1097 (2001). In bullous pemphigoid patientsfollowing therapy with systemic corticosteroids, the frequency of CLA+,but not CLA−, interleukin-13-producing cells is significantly decreased.Decreases in CLA+ cells following corticosteroid treatment is associatedwith clinical improvement. See Teraki, ibid. Neutralization of IL-31 mayimprove clinical outcome of bullous pemohigoid. An IL-31 neutralizingantagonist could be effective in inhibiting, reducing, minimizing orpreventing bullous pemphigoid.

Alopecia Greata

Alopecia greata (AA) is regarded as a tissue-restricted autoimmunedisease of hair follicles in which follicular activity is arrestedbecause of the continued activity of lymphocytic infiltrates. AA resultsin patches of complete hair loss anywhere on the body, though actualloss of hair follicles does not occur, even in hairless lesions.Although clinical signs of inflammation are absent, skin biopsies fromsites of active disease show perifollicular lymphocytic inflammation ofprimarily CD4+ cells, along with a CD8+ intrafollicular infiltrate. SeeKalish R. S. & Gilhar A. J Investig Dermatol Symp Proc: 8, 164 (2003).

Studies have shown that scalp skin infiltrating CD4+ or CD8+ lymphocytesexpress CLA and, in peripheral blood of individuals with AA, the percentof CLA+ CD4+ or CD8+ lymphocytes is significantly higher than that ofnormal controls. Furthermore, patients with severe or progressive AAshow a much higher CLA-positivity compared to patients recovering fromthe disease and a decrease in percent CLA+ cells parallels a goodclinical course. See Yano S., et al., Acta Derm Venereol: 82, 82 (2002).These studies therefore suggest that CLA+ lymphocytes may play animportant role in AA. Xenograft models have demonstrated that activatedT cells are likely to play a role in the pathogenesis of AA. Lesionalscalp from AA patients grafted onto nude mice regrows hair coincidentwith a loss of infiltrating lymphocytes from the graft and, transfer ofactivated lesional T cells to SCID mice can transfer hair loss to humanscalp explants on SCID mice. See Kalish R. S. & Gilhar A. J InvestigDermatol Symp Proc: 8, 164 (2003).

A variety of immunomodulating therapies are part of the usual treatmentfor this disorder however none of these treatments have been consistentin their efficacy. See Tang L., et al., J Invest Dermatol: 120, 400(2003); Tang L., et al., (2004); and Tang L., et al., J Am AcadDermatol: 49, 1013 (2003). Neutralizing anti-IL-31 antibody may beeffective to limit, reduce, inhbit, or prevent the effects of thedevelopment of AA.

Acne Vulgaris/Acne Rosacea

Acne vulgaris, a disorder of the pilosebaceous apparatus, is the mostcommon skin problem of adolescence. Abnormalities in follicularkeratinization are thought to produce the acne lesion. Acne rosacea isdifferentiated from acne vulagaris by the presence of red papules,pustules, cysts and extensive telangiectasias, but the absence ofcomedones (white heads). Increased sebum excretion from sebaceous glandsis a major factor in the pathophysiology of acne vulgaris. Othersebaceous gland functions are also associated with the development ofacne, including sebaceous proinflammatory lipids; different cytokinesproduced locally; periglandular peptides and neuropeptides, such ascorticotrophin-releasing hormone, which is produced by sebocytes; andsubstance P, which is expressed in the nerve endings at the vicinity ofhealthy-looking glands of acne patients. See Zouboulis C. C. ClinDermatol: 22, 360 (2004).

Although the pathophysiology of acne vulgaris and acne rosacea remainsunknown, clinical observations and histopathologic studies suggest thatinflammation of the pilosebaceous follicle may be central to thepathogenesis of rosacea and acne vulgaris. Early studies on analysis ofT cell subsets infiltrating rosacea legions indicated that the majorityof T cells expressed CD4. See Rufli T. & Buchner S. A. Dermatologica:169, 1 (1984).

CD4+ T cells produce IL-31 and IHC analysis of skin for IL-31 expressionsuggests that L-31 is expressed in sebaceous and sweat glands. IL-31stimulation of epidermal keratinocytes induces expression of chemokineswhich likely results in cellular infiltration suggesting that IL-31 maycontribute to the pro-inflammatory response in skin. IL-31 may thereforecontribute to the pathophysiology of acne rosacea and acne vulgaris.Neutralization of IL-31 may improve clinical outcome of acne vulgarisand acne rosacea. An IL-31 neutralizing antagonist could be effective ininhibiting, reducing, minimizing or preventing acne vulgaris and acnerosacea.

Prurigo Nodularis

Prurigo nodularis is an eruption of lichenified or excoriated nodulescaused by intractable pruritus that is difficult to treat. While chronicrubbing results in lichenification, and scratching in linearexcoriations, individuals who pick and gouge at their itchy, irritatedskin tend to produce markedly thickened papules known as prurigonodules. Although prurigo nodularis is not specific to atopicdermatitis, many patients with these nodules also have an atopicreaction, which manifests as allergic rhinitis, asthma, or food allergy.T cells represent the majority of infiltrating cells in prurigo lesionsand these lesions often represents the most pruritic skin lesion inatopy patients.

Topical treatment of prurigo nodularis with capsaicin, an anti-pruriticalkaloid that interferes with the perception of pruritis and pain bydepletion of neuropeptides like substance P in small sensory cutaneousnerves, has proven to be an effective and safe regimen resulting inclearing of the skin lesions. See Stander S., et al., J Am AcadDermatol: 44, 471 (2001). Studies of the itch response in NC/Nga miceusing capsaicin treatment showed that the spontaneous development ofdermatitis lesions was almost completely prevented. Furthermore, theelevation of serum IgE levels was significantly suppressed andinfiltrating eosinophils and mast cell numbers in lesional skin ofcapsaicin treated mice were reduced. See Mihara K., et al., Br JDermatol: 151, 335 (2004). The observations from this group suggest thatscratching behaviour might contribute to the development of dermatitisby enhancing various immunological responses, therefore implying thatprevention of the itch sensation and/or itch-associated scratchingbehaviour might be an effective treatment for AD. See Mihara K., et al.,Br J Dermatol: 151, 335 (2004).

Chronic delivery of IL-31 induces pruritis and alopecia in mice followedby the development of skin lesions resembling dermatitis suggesting thatIL-31 induces itching. See Dillon S. R., et al., Nat Immunol: 5, 752(2004). Neutralization of IL-31 in IL-31 treated mice to preventpruritis and alopecia was tested in Example 10. Neutralization of IL-31may improve clinical outcome of prurigo nodularis. An IL-31 neutralizingantagonist could be effective in inhibiting, reducing, minimizing orpreventing prurigo nodularis.

Skin-Tropic Viruses and Viral Associated Pruritis

Herpes Simplex Virus (HSV)-specific CD8+ T cells in the peripheral bloodand HSV-specific CD8+ T cells recovered from herpes lesions express highlevels of CLA whereas non-skin-tropic herpes virus-specific CD8+ T cellslack CLA expression. See Koelle D. M., et al., J Clin Invest: 110, 537(2002). HSV-2 reactive CD4+ T lymphocytes also express CLA, but atlevels lower than those previously observed for CD8+ T lymphocytes. SeeGonzalez J. C., et al., J Infect Dis: 191, 243 (2005). Pruritis has alsobeen associated with herpes viral infections (See Hung K. Y., et al.,Blood Purif: 16, 147 (1998), though other viral diseases, like HIV, havealso been associated with pruritic skin lesions. Severe, intractablepruritus, often associated with erythematopapular skin lesions andhypereosinophilia, is a condition observed in some nonatopic,HIV-infected patients 36. See Singh F. & Rudikoff D, Am J Clin Dermatol;4, 177 (2003); and Milazzo F., Piconi S., et al., Allergy: 54, 266(1999).

The association of skin-tropic viruses with pruritis and CLA+ T cellssuggests that IL-31 producing T cells may be involved in thepathophysiology of viral infections. Thus, an IL-31 neutralizingantagonist could be effective in inhibiting, reducing, minimizing orpreventing viarl associated pruritis, and neutralization of IL-31 mayimprove clinical outcome of viral associated pruritis.

IL-31 has been shown to induce several chemokine and cytokine genes innormal human epidermal ketatinocytes (NHEKs), including genes encodingGROα, (CXCL1), TARC (CCl17), MIP3β, (CCL19), MDC (CCL22), MIP-3 (CCL23),MIP-1 β (CCL4), and I-309. See Dillon S. R., et al., Nat Immunol: 5, 752(2004). TARC and MDC bind CCR4, a chemokine receptor associated withTh2-type T cells and predominantly expressed by CLA+ T cells inperipheral blood. Both chemokines have been implicated in therecruitment of T cells into the skin of AD patients suggesting thatthese chemokines contribute to the inflammatory process associated withthe pathogenesis of AD. See Example 9 for a model to measure thereduction in TARC and MDC levels in CLA+ T cell mediated disease byadministering an IL-31 antagonist.

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk. Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. IL-31RApolypeptides, soluble heterodimeric and multimeric receptorpolypeptides, or anti-IL-31 antibodies or binding partners of thepresent invention, and the like, could serve as a valuable therapeuticto reduce inflammation and pathological effects in psoriasis, otherinflammatory skin diseases, skin and mucosal allergies, and relateddiseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis, and in some cases rebound, shortly afterstopping immunosuppressive therapy.

Using methods known in the art, and disclosed herein, one of skill couldreadily detect IL-31 in diseases that have a high correlation of CLA+ Tcells. Such methods involve taking a biological sample from a patient,such as blood, saliva, or biopsy, and comparing it to a normal controlsample. Histological, cytological, flow cytometric, biochemical andother methods can be used to determine the relative levels orlocalization of IL-31, or cells expressing IL-31, i.e., monocytes, inthe patient sample compared to the normal control. A change in the level(increase or decrease) of IL-31 expression, or a change in number orlocalization of monocytes (e.g., increase or infiltration of monocyticcells in tissues where they are not normally present) compared to acontrol would be indicative of disease. Such diagnostic methods can alsoinclude measuring TARC and MDC, for example. Such methods are well knownin the art and disclosed herein.

IL-31 polypeptides that bind IL-31RA receptor polypeptides, andantibodies thereto are useful to antagonize or block signaling viaIL-31RA-comprising receptors in the treatment of atopic dermatitis,contact dermatitis, drug induced delayed type cutaneous allergicreactions, toxic epidermal necrolysis, cutaneous T cell lymphoma,bullous pemphigoid, alopecia greata, vitiligo, acne rosacea, prurigonodularis, and Herpes simplex virus.

IL-31 may also be used within diagnostic systems for the detection ofcirculating levels of ligand, and in the detection of diseases that aremediated by CLA+ T cells. IL-31 may also be used within diagnosticsystems for the detection of circulating levels of ligand, and in thedetection of diseases that have a high correlation of CLA+ T cells.Within a related embodiment, antibodies or other agents thatspecifically bind to IL-31 can be used to detect circulating IL-31polypeptides; conversely, IL-31 itself can be used to detect circulatingor locally-acting receptor polypeptides. Elevated or depressed levels ofligand or receptor polypeptides may be indicative of pathologicalconditions, including inflammation and pruritis.

Generally, the dosage of administered IL-31 antibody will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. One skilled in the artcan readily determine such dosages, and adjustments thereto, usingmethods known in the art.

Administration of an anti-IL-31 antibody to a subject can be topical,intradermal, as an inhalant, intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal,by perfusion through a regional catheter, or by direct intralesionalinjection. When administering therapeutic proteins by injection, theadministration may be by continuous infusion or by single or multipleboluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising IL-31 canbe prepared and inhaled with the aid of dry-powder dispersers, liquidaerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235 (1999)). Thisapproach is illustrated by the AERX diabetes management system, which isa hand-held electronic inhaler that delivers aerosolized insulin intothe lungs. Studies have shown that proteins as large as 48,000 kDa havebeen delivered across skin at therapeutic concentrations with the aid oflow-frequency ultrasound, which illustrates the feasibility oftrascutaneous administration (Mitragotri et al., Science 269:850(1995)). Transdermal delivery using electroporation provides anothermeans to administer a molecule having IL-31 binding activity (Potts etal., Pharm. Biotechnol. 10:213 (1997)).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having IL-31 binding activity can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe therapeutic proteins are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having IL-31 binding activity and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having IL-31 binding activity and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates at least aportion of the inflammatory response. Similalry, an agent used to treatitching and pruritis associated with a disease mediated CLA+ T cells, ora disease with a high correlation of CLA+ Tcells, is physiologicallysignificant if its presence alleviates at least a portion of thepruritic or itch response.

A pharmaceutical composition comprising an IL-31 antibody can befurnished in liquid form, in an aerosol, or in solid form. Liquid forms,are illustrated by injectable solutions, aerosols, droplets, topologicalsolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239(1997); Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems,Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer etal., “Protein Delivery with Infusion Pumps,” in Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (PlenumPress 1997); Yewey et al., “Delivery of Proteins from a ControlledRelease Injectable Implant,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)). Othersolid forms include creams, pastes, other topological applications, andthe like.

Polypeptides having IL-31 binding activity can be encapsulated withinliposomes using standard techniques of protein microencapsulation (see,for example, Anderson et al., Infect. Immun. 31:1099 (1981), Anderson etal., Cancer Res. 50:1853 (1990), and Cohen et al., Biochim. Biophys.Acta 1063:95 (1991), Alving et al. “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol. 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,Biochim. Biophys. Acta 1150:9 (1993)).

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Determination of Human Primary T Cell Types thatExpress IL-31 Upon Stimulation

A. Selection of Study Subjects and Biopsies

Twelve patients with AD (moderate to severe disease; median age was 32years old with skin involvement of 5-45%), 6 patients with psoriasis(median age was 56 years old with skin involvement of 10-65%) and 12healthy individuals (median age 34 years) were included in A study afterinformed consent. None of the patients had received any systemiccorticosteroids previously. All patients were off topicalcorticosteroids for one week before their skin biopsy or blood drawing.Two mm punch biopsies were taken from 1) acute erythematous AD lesionsof less than three days' onset, 2) chronic, lichenified AD lesions ofgreater than two weeks' duration, 3) chronic psoriasis lesions, and 4)normal skin. The skin samples were immediately frozen at −70° C. forimmunohistochemistry or Western and immuno-dot blotting.

B. Isolation and Activation of Primary Human T Cell Subsets:

To isolate various T cell subsets, human PBMCs from the donors wereisolated using standard Ficoll gradient centrifugation. Total T cellswere then isolated using the T Cell Isolation Kit II (Miltenyi Biotec)according to the manufacturer's instructions. Separation efficiency wasassessed using standard flow cytometry and determined to be >95% Tcells. To separate CD45RA+ “naïve” T cells from the CD45RO+ “memory” Tcells, the total T cell population was incubated with anti-CD45ROmicrobeads (Miltenyi Biotec) for 15 minutes at +4° C. and magneticallyseparated according to the manufacturers instructions. The naïve andmemory T cell populations were determined to be >90% pure by flowcytometry.

CD45RO+ memory T cells are often tissue specific and cutaneouslymphocyte antigen (CLA) is used to differentiate skin-homing T cellsfrom gut-homing T cells expressing α4/β7 on their surface. To determinewhich of these cell types produce IL-31, CLA+ T cells were isolated fromtotal T cells, activated and conditioned media was collected for theIL-31 bioassay. To do this, total T cells were isolated and thenincubated on ice for 20 minutes in 1 mL of a 1:50 dilution ofanti-CLA-FITC antibody (PharMingen). Cells were then washed, resuspendedin MACS buffer and incubated with anti-FITC microbeads (Miltenyi Biotec)for 15 minutes at +4° C. The cells were then washed, resuspended andmagnetically separated over an LS column according to the manufacturersinstructions. The labeled T cells were later determined to be >80% purewhile the CLA-depleted T cells were >98% CLA−. Both CLA+ and CLA− Tcells were collected and cultured concurrently.

To activate the CD45RA+ and CD45RO+ T cell subsets, cells were culturedovernight in 24-well tissue culture plates pretreated with 2.0 μg/mLanti-CD3 antibody (Southern Biotechnology). The cells were plated at aconcentration of 2.5×10⁶ cells/mL in tissue culture media (RPMI, 5%fetal bovine serum, L-Glutamine and Sodium Pyruvate (all Gibco))supplemented with 2.0 μg/mL anti-CD28 (Southern Biotechnology) andplaced in a +37° C. incubator. After four hours, half of the wells wereharvested, cells pelleted and conditioned media frozen at −20° C. untiltime of IL-31 bioassay.

The CLA+ and CLA− T cell subsets were activated similarly in 48-welltissue culture plates that were pretreated with 2.0 μg/mL anti-CD3antibody (Southern Biotechnology). The cells were activated for 16 hoursor 24 hours in a +37° C. incubator at a concentration of 6.25×10⁵cells/mL. Samples were harvested, cells pelleted and conditioned mediafrozen at −20° C. until time of IL-31 bioassay. For suboptimalactivation, CLA+ T cells were cultured in plates pre-treated with 0.5ug/ml of anti-CD3 antibody.

C. Human IL-31 Bioassay Protocol:

BAF3 cells transfected with hIL-31RA, hOSMRB, and KZ134 (a signaltransducer and activator of transcription-activated luciferase reporter)were grown to 5×10⁵ and 1×10⁶ cells/mL. Cells were washed with assaymedia (RPMI 1640, 10% FBS, L-Glutamine, Sodium Pyruvate, and Pen/Strep(all Gibco)) and resuspended at 3×10⁵ cell/mL in assay medium. In a96-well opaque plate, hIL-31 standards were titered in duplicate from600 pg/mL to 9.38 pg/mL in assay medium via a 100 μL/well, 1:2 serialdilution. Quality control standards were added in duplicate to the plateat 350 pg/mL and 35 pg/mL in 100 μL. Test samples were often diluted 1:2or 1:4 and added in duplicate to the sample wells. 100 μL of the washedBAF3 cells were then added to each well for a final concentration of3×10⁴ cells/well. The plate was then incubated for 16-24 hours at +37°C. in a 5% CO₂ incubator. The plate was then centrifuged at 1200 RPM for5 minutes, media flicked off and 25 μL/well of lysis buffer (Promega)added to each well. After 10 minutes the plate was read on a luminometer(Berthold). The luminometer added 40 μL/well of luciferase substrate mix(Promega) and integrated the luminescence for a period of 4 seconds.Luminescence values were exported to a spreadsheet where they wereanalyzed and converted into picograms of IL-31 per 10⁶ cells per mL ofvolume. The data is summarized in Table 1.

D. Results of IL-31 Bioassay:

The results from the CD45RA+ and the CD45RO+ T cell samples revealedthat IL-31 was primarily produced by activated CD45RO+ memory T cells.The CD45RA+ and CD45RO+ T cells from both donors produced no detectableIL-31 when unstimulated. However, the CD45RO+ samples from both donors#3 and #4 generated significant levels of L-31 following a 24 houractivation with plate-bound anti-CD3 and soluble anti-CD28 (110.4 pg/10⁶cells/mL and 145.6 pg/10⁶ cells/mL respectively). Conversely, when theCD45RA+ T cells from donors #3 and #4 were activated with anti-CD3 andanti-CD28, they produced very low amounts of IL-31 (13.1 pg/10⁶ cells/mLand 12.7 pg/10⁶ cells/mL respectively).

The CLA+ and CLA− T cell samples revealed that IL-31 seems to be madealmost entirely by activated CLA+ T cells. The CLA−population of T cells(which includes naïve T cells, α4/β7 gut-homing memory T cells, andtissue uncommitted T cells) from both donors generated no detectablelevels of IL-31 regardless of time point or activation condition. TheCLA+ T cells on the other hand, generated very high levels of IL-31 whenstimulated with 2.0 μg/mL plate-bound anti-CD3 antibody. Donor #5generated 1385.7 pg/10⁶ cells/mL IL-31 by 16 hours and >1920 pg/10⁶cells/mL by 24 hours. Donor #6 generated 121.3 pg/10⁶ cells/mL IL-31 at16 hours and 328.9 pg/10⁶ cells/mL IL-31 at 24 hours. These resultsclearly demonstrate that of the T cell subsets, IL-31 seems to be madespecifically by cutaneous (CLA+) T cells under standard activationconditions.

TABLE 1 IL-31 IL-31 (pg/10⁶ (pg/10⁶ Donor# Cell Type Activationcells/mL) cells/mL) 6 hr 24 hr 3 CD45RA+ αCD3 + αCD28 Below 13.1Detection 3 CD45RO+ αCD3 + αCD28 8.6 110.4 4 CD45RA+ αCD3 + αCD28 6.712.7 4 CD45RO+ αCD3 + αCD28 11.9 145.6 16 hr 24 hr 5 CLA+ UnstimulatedBelow Below T Cells Detection Detection 5 CLA+ αCD3 1385.7 >1920 T Cells5 CLA− Unstimulated Below Below T Cells Detection Detection 5 CLA− αCD3Below Below T Cells Detection Detection 6 CLA+ Unstimulated Below BelowT Cells Detection Detection 6 CLA+ αCD3 121.3 328.9 T Cells 6 CLA−Unstimulated Below Below T Cells Detection Detection 6 CLA− αCD3 BelowBelow T Cells Detection Detection

Example 2 IL-31 Involvement in Initiation and Perpetuation of ContactHyper-Sensitivity

A. Method I

BALB/c mice are painted on shaved mid-back with 25 ul of 0.5% DNFBdissolved (2,4, dinitro-fluoro-benzene, Sigma, St. Louis Mo.) inacetone:olive oil (4:1) solution using a pipettor. A vehicle controlgroup receives 25 ul of acetone:olive oil only. After 5 days, mice areanaesthetized with isofluorane in an inhalation chamber and both earpinnae of experimental and control animals are measured with anengineer's micrometer (Mitutoyo) to obtain a baseline measurement. Miceare then challenged by applying 10 ul of 0.25% DNFB in acetone:olive oil(4:1) to both sides of each ear of all mice. Contact hyper-sensitivityis measured at 24 h and 48 h later as the difference between the rightear (challenged) and the left ear (unchallenged). All measurements aredone with an engineer's micrometer. Background values are determined bythe difference in ear swelling between the challenged and unchallengedears of naive mice.

Whole blood and serum for FACS and/or ELISA analysis are collected priorto sacrifice and ears are collected for histology.

Method II (Induces Th2 Responses)

BALB/c mice are painted on shaved mid-back with 100 ul of 0.5% FITC(fluorescein isothiocyanate) in a 1:1 solution of acetone/dibutylphthalate (MSDS available using pipettor on days 1, 2 and 8. On day 13,mice are anaesthetized with isofluorane in an inhalation chamber andboth ear pinnae of experimental and control animals are measured with anengineer's micrometer (Mitutoyo) to obtain a baseline measurement. Miceare challenged by applying 25 ul of 0.5% FITC (in 1:1 acetone/dibutylphthalate) to the dorsal surface of each ear. Contact hyper-sensitivityis measured at 24 h and 48 h later as the difference between the rightear (challenged) and the left ear (unchallenged). All measurements aredone with an engineer's micrometer. Background values are determined bythe difference in ear swelling between the challenged and unchallengedears of naive mice. Whole blood and serum for FACS and/or ELISA analysisare collected prior to sacrifice and ears are collected for histology.

Method III (Induces Th1 Responses)

BALB/c mice are painted on shaved mid-back with 25 ul of 2% oxazalone(in 4:1 acetone/olive oil) using pipettor. On day 7, mice areanaesthetized with isofluorane in an inhalation chamber and both earpinnae of experimental and control animals are measured with anengineer's micrometer (Mitutoyo) to obtain a baseline measurement. Miceare challenged by applying 8 ul of oxazalone to the dorsal surface ofeach ear. Contact hyper-sensitivity is measured at 24 h and 48 h lateras the difference between the right ear (challenged) and the left ear(unchallenged). All measurements are done with an engineer's micrometer.Background values are determined by the difference in ear swellingbetween the challenged and unchallenged ears of naive mice. Whole bloodand serum for FACS and/or ELISA analysis are collected prior tosacrifice and ears are collected for histology.

Involvement of IL-31 in the initiation and perpetuation of contacthyper-sensitivity is tested using a neutralizing antibody against IL-31both at the sensitization and challenge phases of the experiment.

Example 3 IL-31 Involvement in Atopic Dermatitis

A. Methods I (Sensitization of NC/Nga Mice)

Male NC/Nga mice were purchased from Charles River Laboratories, Japan.The mice were 4 weeks old on arrival and housed in SPF quarantineconditions for 4 weeks to acclimate. The mice were approximately 10-11weeks old at the start of the antigen sensitization. Mice wereanaesthetized with isofluorane and backs were shaved with electricclippers. Approximately 10 ug of Dermatophagoides pteronyssinus (Dp)(Indoor Biotechnologies, Charlottesville, Va., special order) extractwas injected intradermally at the nape of the neck 3 times per week for5 to 6 weeks until mice developed skin lesions. Control animals received10 ul PBS intradermal injections 3 times per week. The Dp extract wasprepared according to method by Matsuoka and colleagues. Matsuoka H., etal., Allergy: 58, 139 (2003). Briefly, 595 mg Dp lyophilized spentculture extract was dissolved in 12 mL sterile PBS (Gibco). Dp was mixedin a 50 mL Falcon tube on a shaking rocker for 30 minutes. The extractwas spun for 10 minutes at 2000 rpm and the supernatant was collectedand aliquoted into 1 mL cryovial tubes and stored at −20° C.

B. Method II (Sensitization of DO11.10 Mice)

DO11.10 transgenic mice were bred from an in-house colony and werebetween 9.5 and 14 weeks old at start of antigen sensitization. 24 hoursprior to epicutaneous sensitization mice were anaesthetized withisofluorane and the entire trunk (back and abdomen) of mice were shavedwith electric clippers. The mice were then tape stripped with Elastinsurgical tape (Johnson and Johnson) on the back. 1 cm2 sterile gauzepatches were wetted with either 500 ug ovalbumin (Calbiochem 32467) orsterile PBS (Gibco) and adhered to left backside of mice with DuoDermExtra Thin Dressing (ConvaTec 187932). The patch and dressing were thencovered in a body wrap of the Elastin surgical tape so mice could notremove or destroy the patches. Patches were worn for 7 days and removed.The mice were rested for two weeks before having another round ofepicutaneous sensitization. Mice received a total of three one-weeksensitizations.

Results:

Immunohistochemical analysis of IL-31RA expression in lesional andnon-lesional skin from dust mite sensitized NC/Nga and OVA sensitizedDO11.10 animals showed that IL-31RA is expressed by epidermalkeratinocytes in mice, however no significant difference in levels ofexpression was found between antigen sensitized versus PBS sensitizedanimals in this study.

Example 4 IL-31 Involvement Delayed Type Hypersenstitvity

A. Methods

To generate a DTH response, mice were sensitized to antigen on day 0 bysubcutaneous immunization at the base of the tail with 100 ug ovalbumin(OVA) in complete Freund's adjuvant (CFA, 50-100 ul total volume). Oneweek later mice were anesthetized with isofluorane in an inhalationchamber and both ear pinnae of experimental and control animals weremeasured with an engineer's micrometer (Mitutoyo) to obtain a baselinemeasurement. Mice were challenged intradermally with 10 ug OVA in PBS ina total volume of 10 ul into the left ear pinnae, just below the skinwithout hitting any veins. As a control, mice also received an injectionof 10 ul PBS in the right ear pinnae. In some cases, a separate controlgroup given an i.d. injection of OVA in the ear may also be treated withtopical corticosteroids as a positive control to inhibit the reaction.At 24 and 48 hr after challenge, mice were anesthetized and earthickness was measured. Results were expressed as: Specific earswelling=(24 hr measurement−0 hr measurement) for experimental ear—(24hr measurement−0 hr measurement) for negative control ear. Induration,the hallmark of DTH, is detectable by 18 hours after injection ofsensitized antigen and is maximal by 24-48 hours. The lag in the onsetof palpable induration is the reason for naming the response “delayedtype.”

B. Results

IL-31 transgenic mice were tested for DTH, however, due to an increasein ear thickness in un-challenged IL-31 transgenic animals, nostatistically significant difference in DTH could be determined betweenIL-31 Tg animals compared to wildtype controls in this study. IL-31receptor knockout animals were also tested in a DTH response and nosignificant difference in the DTH response could be observed betweenreceptor knockout and wildtype animals.

Example 5 Immunohistochemical (IHC) Staining of IL-31 in Skin Lesionsfrom Uninvolved Psoriatic, and Atopic Dermatitis

Uninvolved psoriatic, atopic dermatitis and normal skin were tested forthe TL-31 ligand by IHC. Positive control cells consisted of BHK cellstransfected with IL-31. Negative controls performed included: (1)un-transfected BHK cells, (2) staining representative tissues and cellswith protein A purified Normal Rabbit serum and detecting antibodybinding as usual. Antibody reagent was E5758 (Rabbit anti-huIL-31 CEE,Aff. Purified at 1.0 mg/ml). Control cells included C02-6020: BHK cellsexpressing zcytor17 Lig hu-CEE/21, and a BHK wild type. Tissues testedincluded acute atopic dermatitis skin samples, chronic atopic dermatitisskin samples, unaffected area skin samples, and normal control skinsamples and other in-house control samples.

The cells and tissues described above were fixed overnight in 10% NBFand embedded in paraffin using standard techniques.

5 μM sections were baked at 61° C. for 30 min for tissue adhesion.Slides were subsequently dewaxed in 3×5′ in xylene and rehydratedthrough graded alcohols as follows: 2×2′ in 100% EtOH, 2×2′ in X95%EtOH, 1×2′ in 70% EtOH. Slides were rinsed in dH20, and then heatinduced epitope retrieval (HIER) was performed for 20 minutes understeam followed by 20 minutes cooling to RT in 10 mM Tris, 1 mM EDTA, pH9.0

Slides were loaded onto a DakoCytomation Autostainer. Slides were rinsedwith TBS/Tween buffer (TBST), prepared as recommend by manufacturer.Endogenous biotin was blocked with a 10 minute incubation in avidinsolution, washed in TBST followed by a 10 minute incubation in biotinsolution. Slides were washed in TBST. A protein block (PBSB) (0.5%Blocking Powder in PBS, Perkin Elmer NEL700001KT.) was applied for 30minutes and rinsed off slides. The primary antibody was diluted to 500ng/ml and was applied for 60 minutes in ChemMate Antibody DilutionBuffer (part#ADB250, Ventana Medical systems).

Tissues washed twice in TBST, and then incubated 45 minutes inbiotinylated Goat anti-Rabbit Ab, 750 ng/ml in PBSB (catalog #BA-1000,Vector Labs). Slides washed twice in TBST. Vectastain Elite ABC Reagent(catalog#PK-7100, Vector Labs) was incubated for 45 minutes. Slideswashed twice in TBST. Signals were developed with DAB+ (catalog#K-3468,DakoCytomation) for minutes at room temperature. Tissue slides were thencounterstained in hematoxylin (catalog#H-3401 Vector Labs), dehydratedand coverslipped in VectorMount (catalog#H-5000, Vector Labs).

Results:

1) Cell Controls:

BHK cells transfected with IL-31 was positively stained with IL-31antibody E5758 while un-transfected cells was negative for thisantibody. The same transfected and un-transfected cells were negativewith anti-rabbit sera.

2) Atopic Dermatitis Skin Analysis:

The staining pattern for IL31 in the AD skin samples is identical tothat of psoriasis skins reported previously: keratinocyte and CD3positive T-cells stained negative for IL31. A weak but rather uniformstaining of the epithelial cells in the secretory portion of the sweatglands was present, but a strong signal was observed in the inner layerof epithelium in the duct portion. Sebaceous gland was positive forIL31. There was no difference in the IL31 staining between AD and normalskin.

Immunohistochemical (IHC) staining of uninvolved psoriatic, atopicdermatitis and normal skin showed strong staining of IL-31 in theholocrine secretion of the sebaceous glands. Considering the phenotypeof IL 31 transgenic mice, it is interesting to note that the sebaceousglands originate as an epithelial bud from the outer root sheath of hairfollicles. In addition to sebaceous glands weak but rather uniformstaining of IL-31 was observed in the epithelial cells in the secretoryportion of the sweat glands and a strong signal in the inner layer ofepithelium was observed in the duct portion of sweat glands.

Example 6 Immunohistochemical (IHC) Staining of IL-31RA in UninvolvedPsoriatic, and Atopic Dermatitis

Uninvolved psoriatic, atopic dermatitis and normal skin were tested forthe IL-31RA by IHC. Positive control cells consisted of BHK cells dualtransfected with IL-31RA and OSMR. Negative controls performed included:(1) un-transfected BHK cells, (2) staining representative tissues andcells with protein A purified Normal Rabbit serum and detecting antibodybinding as usual. Antibody reagent was E6292 (Rabbit anti-huIL-31RAs-CEEv.4 at 1.33 mg/ml). Control cells included C02-5117 BHK cells expressinghuman IL-31RA and human OSMR (Total cells in the pellet: 3.9×106,vitality was >90%) and C04-1587: BHK wild type (Total cells in thepellet: 5×106). Other tissues examined included: 5 Acute atopicdermatitis skin samples, 10 Chronic atopic dermatitis skin samples, 10Unaffected area skin samples, Normal control skin samples, and otherin-house skin samples.

The cells and tissues described above were fixed overnight in 10% NBFand embedded in paraffin using standard techniques.

5 μM sections were baked at 61° C. for 30 min for tissue adhesion.Slides were subsequently dewaxed in 3×5′ in xylene and rehydratedthrough graded alcohols as follows: 2×2′ in 100% EtOH, 2×2′ in X95%EtOH, 1×2′ in 70% EtOH. Slides were rinsed in dH20, and then heatinduced epitope retrieval (HIER) was performed for 20 minutes understeam followed by 20 minutes cooling to RT in 10 mM Tris, 1 mM EDTA, pH9.0

Slides were loaded onto a DakoCytomation Autostainer. Slides were rinsedwith TBS/Tween buffer (TBST), prepared as recommend by manufacturer.Endogenous biotin was blocked with a 10-minute incubation in avidinsolution, washed in TBST followed by a 10-minute incubation in biotinsolution. Slides were washed in TBST. A protein block (PBSB) (0.5%Blocking Powder in PBS, Perkin Elmer NEL700001KT.) was applied for 30minutes and rinsed off slides. Primary antibodies diluted from 665 ng/mlto 1330 ng/ml for IL31RA were applied for 60 minutes in ChemMateAntibody Dilution Buffer (part#ADB250, Ventana Medical systems).

Tissues were washed twice in TBST, and then incubated 45 minutes inbiotinylated Goat anti-Rabbit Ab, 750 ng/ml in PBSB (catalog #BA-1000,Vector Labs). Slides were washed twice in TBST. Vectastain Elite ABCReagent (catalog#PK-7100, Vector Labs) was incubated for 45 minutes.Slides were washed twice in TBST. Signals were developed with DAB+(catalog#K-3468, DakoCytomation) for 10 minutes at room temperature.Tissue slides were then counterstained in hematoxylin (catalog#H-3401Vector Labs), dehydrated and coverslipped in VectorMount(catalog#H-5000, Vector Labs).

Results are shown in Table 2.

TABLE 2 Results of IHC for IL-31RA in skin biopsy specimens frompatients with involved and uninvolved AD compared to healthy volunteersIL-31RA IHC CASE ID SCORE* CD3 IHC SCORE* AD-1 2-3 0-1 AD-2 2-3 2 AD-32-3 1-2 AD-4 3 1 AD-5 2 2 UAD-1 1-2 1 UAD-2 1 0-1 UAD-5 1-2 0-1 UAD-62-3 ND UAD-7 2 1 UAD-8 1 1 UAD-9 1-2 1 UAD-10 2 ND Normal-1 1 0-1Normal-2 0-1 0-1 Normal-3 1 0-1 Abbreviations: AD: atopic dermatitis;UAD: uninvolved AD; ND: Not Done *IHC signal was scored from 0 (nosignal) to 4 (intense signal)

There was a slight up regulation of IL31RA in the epidermis of AD skinsamples. Possibly a small percentage of CD3 positive T-cells werepositive for IL31RA in the AD skins. There were CLA positive cells inall skin samples tested. AD skins may have more CLA positive cells thanthat of the normal or UAD samples.

The receptor for IL-31, IL-31RA was also expressed in the epithelialcells of eccrine sweat glands with the cuboidal epithelial cells in thesecretory portion of the eccrine glands demonstrating slightly higherlevel of IL-31RA protein compared to the duct portion.

Collectively, these data demonstrate that IL-31RA is expressed byepidermal keratinocytes from both control volunteers and AD patients.However, the levels of IL-31RA expressed on keratinocytes from AD skinbiopsies were higher than the levels observed in skin biopsies fromnormal controls, indicating a potential for increased responsiveness toIL-31 in the context of AD.

IL-31RA was also found expressed on a subset of perivascularinfiltrating cells present in skin biopsies from AD patients but was notpresent in control skin biopsies. These IL-31RA+ cells were recognizedby an antibody specific for the tissue macrophage marker CD68,indicating these cells were skin-infiltrating tissue macrophages.

Example 7 Isolation of Skin Infiltrating Cells by Laser CaptureMicroscopy and Analysis of IL-31mRNA by RT-PCR

The presence of skin infiltrating T cells is a distinguishing feature inskin biopsies from AD patients compared to normal individuals. SinceIL-31 is a T cell associated cytokine, the expression of IL-31 inskin-infiltrating T cells in tissue biopsies from AD patients wasexamined. First, the presence of increased numbers of CD3+ T cells inskin tissue biopsies from AD patients compared to normal individuals wasconfirmed by IHC. See Table 2. Next, laser capture microscopy was usedto specifically isolate skin infiltrating cells for analysis of IL-31mRNA by RT-PCR. IL-31 mRNA was expressed by skin infiltrating cells fromacute AD patients. In normal tissues, infiltrating cells are notnormally found and therefore could not be tested. However, the epidermalkeratinocyte layer, which is present in both AD and normal skin, wasanalyzed for IL-31 mRNA expression and lower levels of IL-31 mRNA werefound in normal samples compared to the epidermal keratinocyte layer ofAD samples. Semi-quantitative analysis of IL-31 mRNA expression comparedto an internal control gene (HPRT) showed that although IL-31 mRNAlevels were not significantly different between AD and normal samples,there was a trend towards higher IL-31 expression in skin from ADpatients.

Example 8 IL-31 is Produced by Memory T Cells with a Skin-HomingPhenotype

Analysis of skin biopsies confirmed that the infiltrating CD3+ T cellsin the skin, which express IL-31 mRNA, express the skin-homing markercutaneous lymphocyte antigen (CLA). Of the total T cell population innormal human peripheral blood, IL-31 expression was found to be largelyrestricted to CD45RO+ memory/effector cells as opposed to the CD45RA+naïve T cell population.

In order to determine if IL-31 production was associated with CLA+skin-homing T cells, CLA+ and CLA− T cells were isolated from peripheralblood of patients diagnosed with AD and control volunteers and comparedIL-31 mRNA and protein levels following anti-CD3 plus anti-CD28stimulation. Our results indicate that IL-31 mRNA was significantlyelevated in CLA+ T cells from both AD and normal individuals at both 4 h(p0.0087 and p0.0022 CLA+ compared to CLA− for AD and normal,respectively) and 24 h (p0.0022 CLA+ compared to CLA− for both AD andnormal samples) post stimulation. Analysis of IL-31 protein levels inculture supernatants confirmed that IL-31 was produced predominantly byCLA+ T cells as there was no detectible IL-31 in culture supernatantsfrom CLA− T cells from both AD and control individuals. There were nosignificant differences in L-31 levels between AD and normal patients.We also analysed the production of IL-31 by peripheral blood T cellsthat express other tissue-specific homing markers, such as thegut-specific homing marker α4β7, from normal volunteers. Comparison ofthe IL-31 levels produced by CLA+ T cells and α4β7+ cells demonstratedCLA+ T cells preferentially produce IL-31 compared to the α4β7+ cells(average of 34.5 pg/ml and 14.42 pg/ml IL-31, respectively).

Although both AD patients and normal controls have circulating CLA+ Tcells that express IL-31 upon activation, CLA+ T cells from AD patientsare reported to exist in a more activated state compared to cells fromnormal individuals. Consequently, the threshold of stimulation requiredfor the production of IL-31 by CLA+ T cells may differ betweendermatitis patients and control subjects. To test this hypothesis, westimulated CLA+ T cells from AD patients and control individuals withsub-optimal concentrations of anti-CD3 in the absence of anti-CD28 andanalyzed the production of IL-31 in culture supernatants at 24 h afterstimulation. Our results demonstrate that circulating CLA+ T cells fromsome AD patients produce higher levels of IL-31 compared to cells fromnormal individuals in this study with maximum levels reaching 1200pg/mL, whereas maximal detected levels in normal CLA+ supernatants wasonly 400 pg/ml and maximal detected levels for psorasis patients was 73pg/ml at suboptimal concentrations of anti-CD3 stimulation. Five ofeleven AD patients showed IL-31 levels below the limit of detection ofour assay suggesting there might be a subset of AD patients where IL-31is produced at low levels. This may reflect variations in the stage ofdisease of our study population. Nevertheless, more than half of the ADpatients showed a trend towards higher IL-31 levels compared topsoriasis patients and normal individuals following suboptimalstimulation with anti-CD3. Since more CLA+ T cells are localized in skinof AD patients as compared to normal individuals, our studies suggestthat there is an increased potential for IL-31 activity in the AD skinmicro-environment. Thus, this study may suggest a subpopulation of ADpatients, which have more activated CLA+ T cells producing IL-31.

Example 9 Reduction of TARC and MDC in Response to Anti-Il-31 Antibodyin AD Mouse Models

Method I

Six-week old male NC/Nga mice (CRL Japan) were sensitized intradermallywith 50 μg dust mite extract (D. pteronyssinus, Indoor Biotechnologies)three times a week on the back and scored for AD-like lesions. After 5weeks of sensitization the mice were euthanized and the right ears wereexcised and placed into a single well of a 48-well culture dish(Corning) supplemented with RPMI+2% FBS (GIBCO Invitrogen). Plates wereplaced in 5% CO2 humidity controlled incubators. Supernatants werecollected after 24 hours and frozen at −20° C. until further analysis.

Method II

Twelve-week old female NC/Nga mice (CRL Japan) were sensitizedintradermally with 10 μg SEB (Toxin Technology) in the ear and on theback three times per week. The mice were scored for AD-like lesions.After 5 weeks of sensitization the mice were euthanized and 6 mm biopsypunches were taken from the injected ear of each mouse and placed into asingle well of a 48-well culture dish supplemented with RPMI+2% FBS.Plates were placed in 5% CO2 humidity controlled incubators.Supernatants were collected after 24 hours and frozen at −20° C. untilfurther analysis.

Groups of mice in both studies were treated with either a rat anti-mouseIL-31 monoclonal antibody at 10 mg/kg or vehicle, intraperitoneally twotimes each week starting after 1 to 2 weeks of sensitization.

TARC and MDC concentrations in the 24-hour supernatant samples weremeasured by conventional ELISA (R&D Systems).

TARC and MDC concentrations were lower in ear supernatants fromanti-IL-31 treated mice compared to control mice in both studies,however, these results were not statistically significant when analyzedby ANOVA, probably due to small sample size. When the data from bothexperiments is combined and analyzed there is a statisticallysignificant difference between treated groups.

Example 10 Administration of IL-31 Neutralizing Antibody

Normal female BALB/c mice (CRL) approximately 8 to 12 weeks old wereimplanted subcutaneously with 14-day osmotic pumps (Alzet, #2002)delivering 1 ug/day mIL-31. Groups of mice received intraperitoneal(i.p.) injections of rat anti-mouse IL-31 monoclonal antibody 10 mg/kg(200 ug/mouse) twice weekly starting 1 week prior to IL-31 delivery.Control groups of mice received i.p. injections of vehicle (PBS/0.1%BSA) with the identical dosing schedules. Mice were scored daily foralopecia and pruritis using the following criteria: 0=no scratching,animal appears normal, 1=thinning of coat in small areas, scratchingnoted, 2=minor hair loss (small patches), scratching, 3=moderate hairloss, scratching, and 4=severe hair loss, excessive scratching.

In all experiments, mice treated with rat anti-mIL-31 mAb had a delay inonset of symptoms of approximately 5 to 7 days and a lower overall scorefor alopecia and pruritis. All groups of mAb treated mice (regardless ofdose frequency or concentration) developed alopecia and pruritis similarto control mice by 13 day of the study. These data suggest thatneutralization of IL-31 can delay the onset of the scratch/hairlossresponse induced by IL-31.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

We claim:
 1. A method of treating a skin disorder characterized bycutaneous lymphocyte antigen positive T cells in a mammal comprisingadministering to the mammal a therapeutically effective amount of aneutralizing monoclonal antibody or antibody fragment thereof whichspecifically binds to a polypeptide consisting of amino acid residues27-164 of SEQ ID NO:2, wherein the skin disorder is pruritis associatedwith cutaneous T cell lymphoma, and wherein after administration theskin disorder is improved.
 2. The method of claim 1, wherein theneutralizing monoclonal antibody or antibody fragment is (a) a murinemonoclonal antibody, (b) a humanized antibody derived from (a), (c) achimeric antibody fragment, (d) a human monoclonal antibody; or (e) asingle chain antibody.
 3. The method of claim 1, wherein the antibodyfragment is selected from the group consisting of: a) a Fab fragment; b)a Fab′ fragment; c) a Fv fragments; d) a F(ab′)₂ fragment; and e) asingle chain antibody fragment.
 4. A method of treating a skin disordercharacterized by cutaneous lymphocyte antigen positive T cells in amammal comprising administering to the mammal a therapeuticallyeffective amount of a composition comprising a neutralizing monoclonalantibody or antibody fragment thereof and a pharmaceutically acceptablecarrier, wherein the neutralizing monoclonal antibody or antibodyfragment specifically binds to a polypeptide consisting of amino acidresidues 27-164 of SEQ ID NO:2, wherein the skin disorder is pruritisassociated with cutaneous T cell lymphoma, and wherein afteradministration the skin disorder is improved.
 5. The method of claim 4,wherein the neutralizing monoclonal antibody or antibody fragment is (a)a murine monoclonal antibody, (b) a humanized antibody derived from (a),(c) a chimeric antibody fragment, (d) a human monoclonal antibody; or(e) a single chain antibody.
 6. The method of claim 4, wherein theantibody fragment is selected from the group consisting of: a) a Fabfragment; b) a Fab′ fragment; c) a Fv fragments; d) a F(ab′)₂ fragment;and e) a single chain antibody fragment.